Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease

Marina Cavazzana-Calvo, *123 Salima Hacein-Bey, *123 Geneviève de Saint Basile, 1
Fabian Gross,
2 Eric Yvon, 3 Patrick Nusbaum, 2 Françoise Selz, 1 Christophe Hue,
12 Stéphanie Certain, 1 Jean-Laurent Casanova, 14 Philippe Bousso, 5 Françoise Le Deist,
1 Alain Fischer 124dagger

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gamma c cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gamma c Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gamma c transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

1 INSERM Unit 429,
2 Gene Therapy Laboratory,
3 Cell Therapy Laboratory,
4 Unité d'Immunologie et d'Hématologie Pédiatriques, Hôpital Necker, 75743 Paris Cedex 15, France.
5 INSERM Unit 277, Institut Pasteur, 75730 Paris, France.
*   These authors contributed equally to this work.

dagger    To whom correspondence should be addressed at INSERM Unit 429, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail: fischer@necker.fr


In considering diseases that might be ameliorated by gene therapy, a setting in which a selective advantage is conferred by transgene expression, in association with long-lived transduced cells such as T lymphocytes, may prove critical. SCID-X1 offers a reliable model for gene therapy because it is a lethal condition that is, in many cases, curable by allogeneic bone marrow transplantation (1-4). It is caused by gamma c cytokine receptor deficiency that leads to an early block in T and NK lymphocyte differentiation (1-3). In vitro experiments of gamma c gene transfer have shown that gamma c expression can be restored (5-7), as well as T and NK cell development (8-9), while the immunodeficiency of gamma c- mice can be corrected by ex vivo gamma c gene transfer into hematopoietic precursor cells (10, 11). Long-term expression of human gamma c has also been achieved by retroviral infection of canine bone marrow (12). It has been anticipated that gamma c gene transfer should confer a selective advantage to transduced lymphoid progenitor cells because, upon interaction with interleukin-7 (IL-7) and IL-15, the gamma c cytokine receptor subunit transmits survival and proliferative signals to T and NK lymphocyte progenitors, respectively (2, 3). This hypothesis received further support from the observation that a spontaneously occurring gamma c gene reverse mutation in a T cell precursor in one patient led to a partial, but sustained, correction of the T cell deficiency, including at least 1000 distinct T cell clones (13, 14). Spontaneous correction of the immunodeficiency has otherwise not been observed in several hundred gamma c-deficient SCID patients nor in gamma c- mice (2-4).

Two patients, aged 11 months (P1) and 8 months (P2), with SCID-X1 met the eligibility criteria for an ex vivo gamma c gene therapy trial. SCID-X1 diagnosis was based on blood lymphocyte phenotype determination and findings of gamma c gene mutations resulting either in a tail-less receptor expressed at the membrane (P1) (R289 X) or in a protein truncated from the transmembrane domain that was not expressed at cell surface (P2) (a frameshift causing deletion of exon 6) (15). After marrow harvesting and CD34+ cell separation, 9.8 × 106 and 4.8 × 106 CD34+ cells per kilogram of body weight from P1 and P2, respectively, were preactivated, then infected daily for 3 days with the MFG gamma c vector-containing supernatant (16). CD34+ cells (19 × 106 and 17 × 106/kg, respectively) were infused without prior chemoablation into P1 and P2, ~20 to 40% and 36% of which expressed the gamma c transgene as shown by either semiquantitative PCR analysis (P1) or immunofluorescence (P2). As early as day +15 after infusion, cells carrying the gamma c transgene were detectable by PCR analysis (17) among peripheral blood mononuclear cells. The fraction of positive peripheral blood mononuclear cells increased with time (Fig. 1). T lymphocyte counts increased from day +30 in P1 (who had a low number of autologous T cells before therapy), whereas gamma c-expressing T cells became detectable in the blood of P2 at day +60 (Fig. 2). Subsequently, T cell counts, including CD4+ and CD8+ subsets, increased to 1700/µl from day +120 to +150 and reached values of ~2800/µl after 8 months (Fig. 2). Transgenic gamma c protein expression could not be studied on P1 cells given the presence of the endogenous tail-less protein. However, semi-quantitative PCR performed at day +150 showed that a high proportion of T cells carry and express the gamma c transgene (Fig. 1, A and B). Similar results were observed at day +275. Southern blot analysis of provirus integration in peripheral T cells from both patients revealed a smear indicating that multiple T cell precursors had been infected by the retroviral vector (18).


Fig. 1. gamma c transgene integration and expression. Primers used to detect both PCR and RT PCR products amplify a 904-base pair stretch encompassing the 3' end of the gamma c sequence and downstream vector sequence (5). (A) Semiquantitative PCR analysis of leukocyte subset DNA from P1 and P2. Blood samples were drawn at day +150. T cells (CD3+), B cells (CD19+), monocytes (CD14+), granulocytes (CD15+), and NK cells (CD56+) as well as CD34+ from a bone marrow sample obtained at day +150 from P2 were isolated by a FACStar plus cell sorter (Becton Dickinson) after staining with appropriate mAbs (19). Purity was >99%. Sorted cells were analyzed for the frequency of vector-containing cells (17). Actin DNA was amplified in parallel. Samples from peripheral blood mononuclear cells (PBMC) obtained before treatment are shown as negative controls. A standard curve was constructed by diluting cells containing one copy of the MFG gamma c vector (5) with noninfected cells. All specimens were tested at three dilutions: 1:1, 1:20, and 1:200. (B) Semiquantitative RT-PCR analysis of leukocyte-subset RNA from P1. The same blood sample as in (A) was used. Actin cDNA was amplified in parallel as a control of RNA content. The standard curve was constructed as in (A) (17). No signal was detected in the absence of reverse transcriptase (not shown). Each specimen was diluted to 1:1, 1:500, and 1:5000. [View Larger Version of this Image (33K GIF file)]


Fig. 2. Longitudinal study of lymphocyte subsets from patient 1 (P1) and patient 2 (P2). Absolute counts of T cells (CD3+, CD8+, and CD4+), B cells (CD19+), and NK cells (CD16+, CD56+) are shown as a function of time. Day 0 is the date of treatment. The scale for NK cells is on the right- hand side of each panel. [View Larger Version of this Image (23K GIF file)]

Immunofluorescence studies showed that gamma c was expressed on the membrane of T cells in P2. The magnitude of expression was similar to that of control cells (Fig. 3A), as found in previous in vitro gene transfer experiments (5, 8, 9). These results indicate that sufficient transgene expression had been achieved and that gamma c membrane expression is likely to be regulated by the availability of the other cytokine receptor subunits with which gamma c associates (3). Both alpha beta and gamma delta T cell receptor (TCR)-expressing T cells were detected (Fig. 3B). Polyclonality and Vbeta TCR diversity were demonstrated by using antibodies specific for TCR Vbeta (19) and the immunoscope method (18, 20). In both patients, naïve CD45RA+ T cells were detected, accounting for a majority of the T cell subset (Fig. 3B). In both patients, T cells proliferated from day +105 in the presence of phytohemagglutinin (PHA) and antibodies to CD3 (anti-CD3). The extent of proliferation was the same as that of age-matched controls (Fig. 4A). After primary vaccination, in vitro T cell proliferative responses to tetanus toxoid (P1 and P2: 18,000 and 12,000 cpm, respectively) and polioviruses (P2: 38,000 cpm) were observed within normal range (21). P1 T cells were also found to proliferate in the presence of protein pure derivitive (PPD) (12,000 cpm) as a likely consequence of bacillus Calmette-Guerin (BCG) persistence after immunization at 2 months of age in this immunocompromised child. Five months after cessation of intravenous immunoglobulin (Ig) therapy, antibodies to tetanus and diphtheria toxoids as well as to polioviruses were found in the serum of both patients, together with detectable concentrations of IgG and IgM (Fig. 4B). A normal level of IgA was also detected in the serum of P1. As determined by semi-quantitative PCR and reverse transcriptase-PCR analysis, it was observed that in both cases, a low fraction of B cells carry and express the gamma c transgene (Fig. 1). It is therefore unknown whether antibody responses are provided by untransduced or the few transduced B cells. Residual persistence (< 1%) of administered intravenous immunoglobulins (last given 5 months before measurement of antibody response) could, in part, also contribute. The gamma c-expressing NK cells were detected in the blood of P2 by day 30 (Figs. 1, 2, and 3A). These cells efficiently killed K562 cells in vitro (18). NK cells became detectable in the blood of P1 only from day +150.


Fig. 3. gamma c protein expression and lymphocyte subsets. (A) gamma c protein detection at the surface of lymphocyte subsets from a control and from P2 obtained at day +150. gamma c expression on B cells from P2 after treatment was undetectable (not shown). The y axis depicts the relative cell number, and the x axis shows the logarithm of arbitrary immunofluorescence units. Thin lines are isotype controls; thick lines, staining by the anti-gamma c. Similar results were observed on blood samples obtained at days 275 (P1) and 240 (P2). (B) The percentage of CD45RO+ and CD45RA+ among CD4 and CD8 T cells from P1 and P2 obtained at day +275 and 240, respectively, as well as the percentage of T cells expressing either an alpha beta TCR or a gamma delta TCR. [View Larger Version of this Image (32K GIF file)]


Fig. 4. Functional characteristics of transduced lymphocyte subsets. (A) Longitudinal follow-up of PHA (square , blacksquare )- and anti-CD3 (circle , bullet )-induced proliferation of lymphocytes from P1 (open symbols) and P2 (filled symbols) (8). Background [3H]thymidine uptake was less than 400 cpm. Positive control values are >50 × 103 cpm. (B) Serum immunoglobulin analysis was determined by nephelometry and serum antibody by enzyme-linked immunosorbent assay after immunization (see above). Diphtheria toxoid (Dipht. tox.) was also used for immunization. The last intravenous Ig injections were given at day +90 in both patients. Tet. Tox., tetanus toxoid. Isohemagglutinins to blood group A have now been detected in both patients' sera. [View Larger Version of this Image (13K GIF file)]

As a likely consequence of development and sustained function of the immune system, clinical improvement was observed in both patients. In P2, protracted diarrhea as well as extensive graft-versus-host disease (GVHD)-like skin lesions disappeared. Both patients left protective isolation at days 90 and 95 and are now at home 11 and 10 months, respectively, after gene transfer without any treatment. Both enjoy normal growth and psychomotor development. No side effects have been noted. A similar result has since been achieved in a third patient 4 months after gene transfer (22). These results demonstrate that in these patients, a selective advantage was conferred to T and NK lymphocyte progenitors, enabling full-blown development of mature and functioning T and NK lymphocytes (23).

These overall positive results contrast with the failure of previous attempts to perform ex vivo gene therapy in adenosine deaminase (ADA)-deficient patients (24-27). Concomitant administration of ADA enzyme to these patients is likely to have counterbalanced the potential growth advantage of the transduced cells in this setting (23). Also, advances in the methodology of gene transfer into CD34+ cells, i.e., the use of a fibronectin fragment (28) as well as of a cytokine combination enabling potent CD34 cell proliferation, contributed to the success of gamma c gene therapy.

Because gamma c gene transfer was achieved without any additional myeloablative or immunosuppressive therapy, these results pave the way for a possible extension of this therapeutic approach to other genetic diseases characterized by defective cell-subset generation, such as other forms of SCID (29). The kinetics of T cell development in gamma c gene transfer is similar to that observed in SCID patient recipients of haploidentical stem cell transplantation (4), suggesting that early progenitor cells have been infected by the MFG gamma c virus and effectively transduced. The hypothesis that transduced autologous T cells in P1 account for the development of the T cell compartment is unlikely because (i) the infected CD34+ cell population was contaminated by less than 0.1% CD3+ T cells; (ii) a thymic gland (27 mm by 25 mm by 25 mm at day +275) became detectable by ultrasound echography, indicative of thymopoiesis, whereas most T cells at day +275 exhibit a naïve CD45RA+ phenotype; and (iii) the T cell repertoire was polyclonal and diverse. In both patients, it was shown that at day +150, a fraction of bone marrow CD34+ cells harbored and expressed the gamma c transgene (Fig. 1, P2). It was not possible to determine whether more primitive cells, i.e., CD34+CD38- cells, were transduced because of insufficient bone marrow sample. In the mouse, a common lymphoid progenitor (CLP) gives rise to the different lymphocyte populations (30). If a human counterpart of CLP exists, it would be the best candidate from among the earliest cells that were transduced ex vivo from these patients. Identification of integration sites in the various cell lineages could help determine the permissive differentiation stage. The question of the persistence of T and NK cell generation has yet to be addressed. If infected cells have no self-renewal capacity and have a short life-span, new generation of T and NK cells should cease. However, the fact that a thymic gland is still detectable 9 months after gamma c gene transfer suggests that thymopoiesis is still ongoing. Follow-up of the SCID-X1 patient in whom a spontaneous reversion mutation occurred in a T cell precursor (13, 14) indicates that gene transfer could be sufficient to provide a functional memory T cell pool for a number of years. This optimistic view will require careful sequential appraisal. Kohn et al. have previously shown that transgenes placed under the control of the long-terminal repeat (LTR) viral promoter can be silenced in quiescent T cells (31). Although the identification of silencing sequences in the MFG LTR makes this a strong possibility (31), down-regulation of gamma c expression has not been observed so far in these two patients, in gamma c-deficient mice treated by ex vivo gamma c gene transfer (11), or in cell lines maintained in culture over 1 year (5).

Follow-up will be required to assess the long-term effects of ex vivo gamma c gene transfer in CD34+ cells of SCID-X1 patients. To date, this methodology has resulted in the sustained correction (up to 10 months) of the SCID-X1 phenotype in two patients, including a patient in whom the mutated protein is expressed at the cell surface. It is presumed that the effect results from a strong positive selective pressure provided to the corrected lymphoid progenitors.

REFERENCES AND NOTES

  1. M. Noguchi, et al., Cell 73, 147 (1993) [ISI] [Medline] .
  2. K. Sugamura, et al., Annu. Rev. Immunol. 14, 179 (1996) [ISI] [Abstract/Full Text] .
  3. W. J. Leonard. Annu. Rev. Med. 47, 229 (1996).
  4. R. H. Buckley, et al., N. Engl. J. Med. 340, 508 (1999) [ISI] [Medline] .
  5. S. Hacein-Bey, et al., Blood 87, 3108 (1996) [ISI] [Abstract] .
  6. F. Candotti, et al., Blood 87, 3097 (1996) [ISI] [Abstract] .
  7. N. Taylor, et al., Blood 87, 3103 (1996) [ISI] [Abstract] .
  8. M. Cavazzana-Calvo, et al., Blood 88, 3901 (1996) [ISI] [Abstract] .
  9. S. Hacein-Bey, et al., Blood 92, 4090 (1998) [ISI] [Abstract/Full Text] .
  10. M. Lo, et al., Blood 94, 3027 (1999) [ISI] [Abstract/Full Text] .
  11. C. Soudais, et al., Blood 95, 3071 (2000) .
  12. T. Whitwam, et al., Blood 92, 1565 (1998) [ISI] [Abstract/Full Text] .
  13. V. Stephan, et al., N Engl. J. Med. 335, 1563 (1996) [ISI] [Medline] .
  14. P. Bousso, et al., Proc. Natl. Acad. Sci. U.S.A. 97, 274 (2000) [ISI] [Abstract/Full Text] .
  15. Patient 1 had pneumocystis carinii pneumonitis and had received BCG immunization. Patient 2 suffered from recurrent oral candidiasis, pneumocystis carinii infection, protracted diarrhea, failure to thrive, and GVHD-like skin lesions. Neither patient had an HLA (human leukocyte antigen)-identical sibling. Patients were placed in a sterile isolation ward and received nonabsorbable oral antibiotics and intravenous Igs every 3 weeks for 3 months. Parents gave informed consent for participation in the trial.
  16. The defective MFG gamma c vector has been described previously (5). It was packaged in the psi crip cell line. The MFG gamma c vector-containing supernatant was manufactured and provided by Genopoietic (Lyon, France) under GMP guidelines. The vector supernatant was free of replication-competent retrovirus as determined by S+L- assay and a beta -galactosidase mobilization test [ R. H. Bassin, N. Tuttle, P. J. Fischinger, J. Cancer 6, 95 (1970) ; M. Printz, et al., Gene Ther. 2, 143 (1995) [ISI] [Medline] ]. Concentration of the virus in the supernatant was 5 × 105 infectious virus particles (5). Marrow CD34+ cells were positively selected by an immunomagnetic procedure (CliniMACS, Miltenyi Biotec, Bergish Gladbach, Germany). CD34 cells were cultured in gas-permeable stem cell culture (PL-2417) containers (Nexell Therapeutics, Irvine, CA), at a concentration of 0.5 × 106 cells/ml in X-vivo 10 medium (Biowhittaker, Walkerville, MD) containing 4% fetal cell serum (Stem Cell Technologies, Vancouver, Canada), stem cell factor (300 ng/ml, Amgen), polyethylene glycol-megabaryocyte differentiation factor (100 ng/ml, Amgen), IL-3 (60 ng/ml, Novartis), and Flt3-L (300 ng/ml, R&D Systems, Minneapolis, MN) for 24 hours at 37°C in 5% CO2. Containers were precoated with the CH296 human fragment of fibronectin (50 µg/ml) (TaKaRa, Shiga, Japan). Retroviral containing supernatant was added every day for 3 days. Cells were then harvested, washed twice, and infused back into the patients.
  17. For semiquantitative PCR and RT-PCR analysis, DNA was isolated from the indicated cell populations. A reference standard curve was constructed by diluting cells from a SCID-X1-derived Epstein-Barr virus (EBV)-B cell line containing one copy per cell of the MFG gamma c provirus (5) in uninfected cells from the same EBV-B cell line (100, 10, 1, 0.1, 0.01, and 0.001%). DNA from each sample was also quantified by actin gel amplification. MFG gamma c primers sequences and actin primers sequences are available on request. DNA was amplified in a 50 µl of PCR reaction mixture by using 30 cycles at an annealing temperature of 60°, for gamma c primers and 68°C for actin primers. A sample of the amplified product was separated on a 1% agarose gel and analyzed by ethidium bromide staining. RNA was prepared with the RNA easy kit (Qiagen) and was reverse-transcribed with the Superscript Preamplification System (Gibco-BRL). gamma c proviral and beta -actin cDNA amplification were performed as described above. Quantification of expression was made by comparison with RNA isolated from the same standard curve of diluted cells.
  18. M. Cavazzana-Calvo et al., data not shown.
  19. The following monoclonal antibodies (mAbs) were used in immunofluorescence studies: anti-gamma c chain: Tugh 4 (rat IgG2, PharMingen, San Diego, CA); anti-CD3: Leu 4 (IgG2a, Becton Dickinson, San Diego, CA); anti-CD4: Leu3a (IgG1, Becton Dickinson); anti-CD8: Leu 2a (IgG1, Becton Dickinson); anti-CD19: J4 119 (IgG1 Immunotech, Marseille, France); anti-CD14: Leu M3 (Becton Dickinson); anti-CD16: 3G8 (IgG1, Immunotech); anti-CD56: MY31 (IgG1, Becton Dickinson); anti-CD15 (IgM, PharMingen); anti-TCR alpha beta : BMA031 (IgG1, Immunotech); anti-TCR gamma delta : IMMU 515 (IgG1, Immunotech); anti-CD45RO: UCHL1 (IgG2a, Immunotech); anti-CD45RA: 2H4 (IgG1, Coulter Clone, Margency, France); anti-CD34: HPCA-2 (IgG1, Becton Dickinson); anti-TcR Vbeta 2: MPB2D5 (IgG1, Immunotech); anti-TcR Vbeta 3: CH92 (IgM, Immunotech); anti-TcR Vbeta 5.1: IMMU 157 (IgG2a, Immunotech); anti-TcR Vbeta 5.2: 36213 (IgG1, Immunotech); anti-TcR Vbeta 5.3: 3D11 (IgG1, Immunotech); anti-TcR Vbeta 8: 56C5.2 (IgG2a, Immunotech); anti-TcR Vbeta 9: FIN9 (IgG2a, Immunotech); anti-TcR Vbeta 13.1: IMMU 222 (IgG2, Immunotech); anti-TcR Vbeta 13.6: JU74.3 (IgG1, Immunotech); anti-TcR Vbeta 14: CAS1.13 (IgG1, Immunotech); anti-TcR Vbeta 17: E17.5F3.15.13 (IgG1, Immunotech); anti-TcR Vbeta 21.3: IG125 (IgG2, Immunotech). Fluorescence staining was done with phycoerythrin- or fluorescein isothiocyanate-conjugated mAbs. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson).
  20. C. Pannetier, et al., Proc. Natl. Acad. Sci. U.S.A. 90, 4319 (1993) [ISI] [Abstract] .
  21. Unstimulated lymphocyte proliferations were <1000 cpm. Control positive values of antigen-stimulated proliferations were >10,000 cpm.
  22. This patient was treated at 1 month of age. Within 3 months, T and NK lymphocyte counts reached age-matched control values. The gamma c expression at T and NK cell surfaces was fully restored. The child is at home without any therapy, 4 months after treatment.
  23. C. Bordignon, Nature Med. 4, 19 (1998) [ISI] [Medline] .
  24. C. Bordignon, et al., Science 270, 470 (1995) [ISI] [Abstract] .
  25. D. B. Kohn, et al., Nature Med. 1, 1017 (1995) [ISI] [Medline] .
  26. D. B. Kohn, et al., Nature Med. 4, 775 (1998) [ISI] [Medline] .
  27. V. W. Van Beusechem, et al., Gene Ther. 3, 179 (1996) [Medline] .
  28. H. Hannenberg, et al., Nature Med. 2, 876 (1996) [Medline] .
  29. A. Fischer and B. Malissen, Science 280, 237 (1998) [ISI] [Abstract/Full Text] .
  30. M. Kondo, I. L. Weissman, K. Akashi, Cell 9, 661 (1997) .
  31. P. B. Robbins, et al., Proc. Natl. Acad. Sci. U.S.A. 95, 10182 (1998) [ISI] [Abstract/Full Text] .
  32. We thank the medical and nursing staff of the Unité d'Immunologie et d'Hématologie pédiatriques, Hôpital des Enfants-Malades, for patient care. We also thank C. Harré and C. Jacques for technical help; D. Bresson for preparation of the manuscript; N. Wulfraat for patient referral; O. Danos, M. Fougereau, P. Mannoni, C. Eaves, and L. Coulombel for advice; A. Gennery for assistance with English translation; B. Bussière, C. Cailliot, and J. Caraux (Amgen, France) for providing SCF and MGDF; J. Bender and D. Van Epps (Nexell Therapeutics, Irvine, CA) for providing containers; and S. Yoshimura and I. Kato (Takara Shuzo, Shiga, Japan) for providing the CH-296 fibronectin fragment. Supported by grants from INSERM, Association Française des Myopathies, Agence Française du Sang, and the Programme Hospitalier de Recherche Clinique (Health Ministry).
28 December 1999; accepted 10 March 2000



Volume 288, Number 5466, Issue of 28 Apr 2000, pp. 669-672.
Copyright © 2000 by The American Association for the Advancement of Science.